One becomes three: An integrative morphological and molecular analysis of the windowpane oyster Placuna (Bivalvia: Pectinida) reveals new species

Abstract For decades, many marine animals have been considered to exhibit cosmopolitan or transoceanic distribution. This situation is prevalent in Asia, where many species were collected and named by American or European experts in the 1700s to early 1900s. Using the windowpane oysters Placuna—a small genus of bivalves with five recognized species—we show that careful analysis is required to reassess the validity of these species. Currently, only two species of Placuna (P. placenta and P. ephippium) widely reported in the Indo‐Pacific region have been recorded from Chinese coastal waters. Here, we described two new species of Placuna from China. Placuna vitream sp. nov. can be distinguished from P. placenta by its larger ridge angle. Phylogenetic analysis using five gene fragments fully supported that P. vitream sp. nov. is a sister to the specimen from Singapore identified as P. placenta and more distant from other Placuna species with available molecular data. Besides, based on subfossil shells, we describe Placuna aestuaria sp. nov. that differs from its congeneric species by its broad hinge, medium ridge angle, and nearly straight ridges. Finally, we suggest a combination of hinge structure and ridge angle that can be used for identifying Placuna species and preparing a key to this genus. Our findings of two new species expand the diversity of Placuna and prompt reassessment of the many presumably widely distributed marine species in Asia.

larval dispersal period observed in certain taxa (Hansen, 1980;Scheltema, 1971;Schulze et al., 2012).Reassessment of such widely distributed species, however, has shown mixed results.While some have discovered morphologically similar cryptic species such as those from chemosynthetic habitats (Bickford et al., 2007;Hutchings & Kupriyanova, 2018;Pérez-Portela et al., 2013;Wang et al., 2020), others have confirmed their wide distribution patterns (McCowin et al., 2019;Thomas et al., 2020).Nevertheless, a significant number of marine species have not undergone a thorough evaluation of their species identity and distribution, impeding our comprehension of diversity and biogeographical patterns.
The family Placunidae Rafinesque, 1815, commonly called windowpane oysters, windowpane shells, or capiz shells, serve as a compelling case for investigating species identity and distribution patterns.This family, classified in the order Pectinida Gray, 1854, inhabits predominantly the coastal waters of the Indo-Pacific.The family is monogeneric, with Placuna Lightfoot, 1786 as the only genus, and it contains five extant species (P.ephippium Retzius, 1788, P. lincolnii Gray, 1849, P. lobata Sowerby, 1871, P. placenta Linnaeus, 1758, and P. quadrangula Retzius, 1788) and two fossil species (P.mandirantjanensis Martin, 1909 andP. pseudoplacenta Martin, 1909), all named between 1758 and 1871 (MolluscaBase, 2024).The type species P. placenta, characterized by semitransparent shells, has high commercial value: it is an edible species and its shells are widely used for crafting ornamental wares and traditional windowpanes (Gallardo et al., 1995;Rustia et al., 2023).This species is widely reported from the northern to the eastern Indian Ocean and the western to southern Pacific Ocean (MolluscaBase, 2024).In China, P. placenta has been reported from the intertidal zone of the northern South China Sea and the southern East China Sea (Li et al., 2019;Liu, 2008).However, our preliminary analysis of the mitochondrial cytochrome c oxidase subunit I (cox1) of "P.placenta" specimens from the Chinese coastal waters revealed Kimura 2-parameter (K2P) genetic distances >11% with a P. placenta sample collected from Singapore (Bieler et al., 2014).These K2P distances are much larger than the variations typically considered intraspecific for bivalves (i.e., 2.0%) (Lin et al., 2022;Yu & Li, 2012).In addition, we collected several shells of 8000-6000 years old from Hong Kong (WWF Hong Kong, 2013), which appear to come from an extinct species since we cannot find any living windowpane oysters in that area.These live specimens and subfossil shells appear distinct from each other and the recognized species of the genus.
Therefore, this study aims to characterize the two new species of Placuna from China based on a rigorous molecular phylogenetic framework and morphological analysis.We describe the morphology of the two new species of Placuna and construct an identification key to all species of the genus.For species with soft tissues available, we amplified five gene fragments and conducted a phylogenetic study of Placuna spp.Our results enrich the genetic information of Placunidae and prompt reevaluation of marine bivalve species that are considered widely distributed (Jackson et al., 2015).

| Other materials studied
More specimens of P. vitream sp.nov.were purchased from the fisherman in Dongmen Market, Haikou, Hainan Island, China, in May 2023, while an adductor muscle sample of P. vitream sp.nov.was collected from Xiajin Bay (24°30.48'N, 118°12.25′E), Xiamen, Fujian, China, in January 2023.The specimens of P. ephippium were collected from the same location as the P. vitream sp.nov.type specimens.The samples of P. quadrangula were collected from the intertidal zone of Bilangbilangan Island (10°14.49'N, 124°27.14′E), Philippines (Table 1).

TA B L E 1
Specimens used in this study.

| Morphological measurement and photography
The Placuna shells were measured using a vernier caliper to determine the shell length (L), height (H), hinge length (HL), hinge height (HH), anterior hinge length (AHL), anterior ridges length (ARL), posterior ridges length (PRL), scar length (SL), anterior length (AL), dorsal height (DH), and ridge angle (RA) (Figure 1).The specimens were photographed using an EOS 5D Mark IV camera (Canon, Japan), and their details were observed using a digital Stereo Microscope MZ1270i Imaging System (Nikon, Japan).The type specimens used in this study were deposited in the Tropical Marine Biodiversity Collections of the South China Sea (TMBC), Chinese Academy of Sciences, Guangzhou, China.

| DNA extraction and PCR
The genomic DNA of P. vitream sp.nov., P. ephippium, and P. quadrangula was extracted from the adductor muscle using the CTAB method (Stewart & Via, 1993).The genomic DNA quality was determined using agarose gel (1.0%) electrophoresis and quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, USA).
The PCR products were bidirectionally sequenced on an ABI PRISM 3730xl DNA Analyzer (Thermo Fisher Scientific).The sequences for phylogenetic analyses were assembled using SeqMan (DNASTAR).

| Distribution
Currently known from Xincun Port, Sanya, and Xiajin Bay, Xiamen in China.

| Etymology
The epithet "vitream" refers to this species' translucent and pearl-like glittery shells.

| Diagnosis
Shell up to 110 mm, subcircular and translucent.Hinge slim with clear hinge teeth.Umbones slightly prominent, close to the anterior end of the hinge.Auricles obvious, anterior auricle larger than posterior auricle.Ridges angle moderate from 28° to 31°.
Hinge ridges and ligaments slightly curved; anterior ridge shorter than posterior ridge.

| Description
Shell (Figures 2 and 3a,b, Tables 3 and 4) subcircular, translucent, and very compressed.Left valve more convex than right valve.
External surface with growth lines, mauve dorsal, and gray lamella,

| Remarks
Within the genus Placuna, P. vitream sp.nov.can be distinguished from P. ephippium, P. quadrangula, P. lincolnii, and P. lobata by its unequal length of the hinge ridges and ligaments (Das et al., 2019;Dunker, 1879;Matsukuma, 1987) (Figures 3a,b and 6, Tables 3 and   4).The AHL/HL ratio of P. vitream sp.nov. is much smaller than that of P. ephippium and P. quadrangula, which means the umbones of the former are located at the anterior end of the hinge, whereas those of the latter two are located near the hinge center.
Although such data are unavailable for P. lincolnii and P. lobata, previous studies showed that their umbones are in the middle of the hinge (Dunker, 1879;Gray, 1849;Matsukuma, 1987).Placuna vitream sp.nov.and two fossil species P. pseudoplacenta and P. mandirantjanensis exhibit similar shell shapes, outlines, and V-shaped hinges.However, the ridge angles of the two fossil species are substantially larger (RA > 60°) (Martin, 1909) 3 and 4   similar to the windowpane shell P. placenta, which may explain its records as a common species in Chinese coastal waters (Li et al., 2019;Liu, 2008).They are extremely similar in shell shape, outline, hinge ridge and ligament form, and autonomy (Yonge, 1977).
Considering the widely distributed so-called "P.placenta" and the taxonomic uncertainty of the specimens from other locations, we only compared P. vitream sp.nov.with the holotype of P. placenta (Figure 3f) stored in the Linnean Society of London (https:// linne an-online.org/ ).The P. placenta holotype, whose sampling locality is unknown as Carl Linnaeus only wrote "Pelago" = the Ocean for its habitat, processes a gentle dorsal outline and inconspicuous umbones (Linnaeus, 1758).Our statistical study shows that ridge angle is a significant feature for species identification within Placuna (Figure 3g).The ridge angle of P. placenta is about 21° (Linnaeus, 1758) 4).The Placuna species form a single clade of Placunidae with new data from three species: P. quadrangula, P. ephippium, and the new species.Among them, the P. vitream sp.nov.The intraspecific K2P genetic distances within P. vitream sp.
The large interspecific genetic distances of cox1 between P. vitream sp.nov.and other Placuna species support our recognition of the new species.Carmen K. M. Or, in July 2023.

| Distribution
Currently known only from the Mai Po Nature Reserve, Hong Kong SAR, China.

| Etymology
The species epithet "aestuaria" comes from "estuarial" in Latin, which refers to the estuarine waters of the type locality of this species.

F I G U R E 5
The Kimura 2-parameter (K2P) genetic distances (%) based on the cox1 fragments among Pectinida.The analysis was performed based on a 615-bp matrix, with the species Lima lima as the outgroup.The sequences P. vitream sp.nov.HT1-5 represent the specimens TMBC031019-TMBC031023 collected from the type locality, while the sequences P. vitream sp.nov.PT1-2 represent the specimens collected from Haikou and Xiamen, respectively.
the AHL/HL and HH/HL ratios), ridge angle, and auricle features to distinguish the species of Placuna (Figure 3g).
Molecular data are also very limited in Placunidae.Prior to this study, only five gene fragments from P. placenta and a cox1 sequence of an undetermined Placuna species have been published (Bieler et al., 2014;Chang et al., 2020;Sharma et al., 2013).The lack of molecular data makes it difficult to determine the phylogenetic relationships among Placuna spp.and the position of Placuna in Pectinida.
The two mitochondrial and three nuclear gene fragments for P. ephippium, P. quadrangula, and P. vitream sp.nov.generated in this study will be useful for future phylogenetic studies of Placula and even Bivalvia.Furthermore, given that DNA-based analyses have been widely used to distinguish morphologically similar species, such as species in Mytilida, Pteriida, and Venerida (Lemer et al., 2014;Ni et al., 2012;Shen et al., 2014), our discovery of P. vitream sp.
nov. suggests that Placuna might be more diverse than previously thought, and "P.placenta" specimens from other locations should be examined to determine their real identities (Gallardo et al., 1995;Li et al., 2019;Rustia et al., 2023;Song et al., 2022).
without radial lines.Length up to 110 mm, nearly equal to height (L/H = 0.960-1.050)and approximate equilateral (AL/L = 0.450-0.507).Umbones slightly prominent, close to anterior end of hinge (AHL/HL = 0.205-0.325).Auricles obvious, anterior auricle larger than posterior auricle.Hinge slim (HH/HL = 0.082-0.135),slightly rounded, with obscure hinge teeth or without teeth.Hinge ridges and ligaments slightly curved, inverted V-shaped, with moderate ridge angle (RA) from 28° to 31°.Anterior hinge ridge and ligament shorter than posterior ridge (ARL/PRL = 0.594-0.761).Anterior F I G U R E 2 External and internal views of the left and right valves from three pairs of Placuna vitream sp.nov.specimens.(a-d) External view of left valve, external view of right valve, internal view of left valve, internal view of right valve of the holotype (TMBC031019), respectively; (e-h) External view of left valve, external view of right valve, internal view of left valve, internal view of right valve of the paratype 1 (TMBC031020), respectively; (i-l) External view of left valve, external view of right valve, internal view of left valve, internal view of right valve of the paratype 2 (TMBC031021), respectively.Scale bar: 10 mm.pedal retractor scar oval, close to middle between ligament posterior ends.Adductor muscle scar subcircular, close to valve center.Anatomy (Figure 3c): Mantle large, thin, and semitransparent, with distinct mantle edge and row of tentacles at the margin.Large and Cshaped gill at anterior side.Digestive diverticula subcircular.Adductor muscle large, circular, near shell center.Foot small, anteriorly located between digestive diverticula and adductor muscle.Ventricle circular, posteriorly located between digestive diverticula and adductor muscle.Gonad folded scrotiform, posterior to digestive diverticula.
than P. vitream sp.nov.(28°-31°), and the anterior hinge ridge of P. vitream sp.nov. is straighter.Notably, P. vitream sp.nov. is morphologically most F I G U R E 3 Internal views of three Placuna species and the statistical study of morphological features of the two new species.(a,b) The left and right valves of P. vitream sp.nov.holotype (TMBC031019); (c) Anatomy of P. vitream sp.nov.paratype 2 (TMBC031021); (d,e) The left and right valves of P. aestuaria sp.nov.holotype (TMBC031038).(f) The type specimen of Placuna placenta stored in the Linnean Society of London (https:// linne an-online.org/ ) for morphological comparison.(g) Statistical study of the shell morphological indices and ridge angles between two new species.The details are shown in Tables specimens are fully supported to be sister to the specimen from Singapore identified as P. placenta (posterior probability = 1.0, bootstrap value = 100).Among the eight families of Pectinida included in the analyses, only Anomiidae is paraphyletic, with Anomia simplex and Anomia sp.being sister to Placunidae, and together they are sister to A. chinensis and a clade comprising A. ephippium, Pododesmus caelata, and P. patellifomis.F I G U R E 4 Phylogenetic relationships of Pectinida.(a) The placement of Placuna vitream sp.nov. in Pectinida revealed by a 4745-bp concatenated alignment (cox1-16S rRNA-18S rRNA-28S rRNA-histone H3) using Bayesian inference (BI) analysis.Bootstrap values from maximum-likelihood analysis and posterior probabilities values from BI analysis are given at nodes.Sequences generated from this study are highlighted in red color.The sequences P. vitream sp.nov.HT1-5 represent the specimens TMBC031019-TMBC031023 collected from the type locality, while the sequences P. vitream sp.nov.PT1-2 represent the specimens collected from Haikou and Xiamen, respectively.
2b.Shell oval to subcircular……P.lincolnii.3a.Shell flexuous with purple or brown color inter surface……P.ephippium.3b.Outer surface ornamented with ribs……P.lobata.3c.Shell semitransparent with radial reddish-brown stripes……P.quadrangula.4a.Ridges gently diverged with an angle less than 40°……5.4b.Anterior ridge strongly curved and ridge angle 60°or larger……7.F I G U R E 7 External and internal views of the left and right valves from three pairs of Placuna aestuaria sp.nov.specimens.(a-d) External view of left valve, external view of right valve, internal view of left valve, internal view of right valve of the holotype (TMBC031038), respectively; (e-h) External view of left valve, external view of right valve, internal view of left valve, internal view of right valve of the paratype 1 (TMBC031039), respectively; (i-l) External view of left valve, external view of right valve, internal view of left valve, internal view of right valve of the paratype 2 (TMBC031040), respectively.Scale bar: 10 mm.
Type specimens of P. vitream sp.nov.werecollectedfrom the intertidal zone of Xincun Port (18°24.55'N,109°58.49′E),Sanya,Hainan Island, China, in November 2023.The type specimens of P. aestuaria sp.nov.(emptyshellsonly) were collected from the Mai Po Nature Reserve (22°29.03'N,114°01.56′E),HongKong, in July 2023, buried in the unearthed mud located in the Deep Bay area.The adductor muscles of fresh specimens were preserved using 100% ethanol for DNA extraction, and all shells were cleaned and kept in room temperature (Table1).
Measurements of the left valves of the specimens (in mm where applicable).
TA B L E 4 , which is slightly smaller than 28° to 31° in P. vitream sp.nov.(Figures2 and 3a,b, Table2).In conclusion, P. vitream sp.nov.can be distinguished from its congeneric species and regarded as a new species.